Afamin-containing compositions and methods of use

ABSTRACT

The use of Afamin, in particular in combination with vitamin E is described for producing a preparation for the treatment of oxidative stress.

This application is a U.S. national phase application under 35 U.S.C. §371 of International Application No. PCT/AT02/00132 filed 30 Apr. 2002,which claims priority to Austrian Application No. A 696/2001 filed 30Apr. 2001.

The invention relates to preparations of vitamin E in combination withAfamin.

Afamin is a 87 kDa protein which belongs to the albumin group andstructurally and biochemically has many things in common with theproteins of this group, i.e. with human serum albumin, humanα-fetoprotein or human vitamin D binding protein. Afamin has alreadybeen cloned and sequenced and thus is also available in recombinant form(WO 95/27059).

Apart from its sequence homologies, little is known about the functionof Afamin. The possibility has been discussed that Afamin has sterolebinding sites, yet probably does not bind actin. Due to an existing, yetnot overwhelming similarity between Afamin and albumin, it is doubtedthat these proteins bind the same ligands (Lichenstein et al., TheJournal of Biological Chemistry, 269 (27) (1994), pp. 18149-18154).

Biochemical and physiological examinations have shown that Afamin hasvitamin E-binding properties and does not only occur in blood, but alsoin other body or organ fluids, such as, e.g., cerebrospinal, follicularand seminal fluid. The use of Afamin for determining the fertility ofmammals has been described in WO 01/01148 A1.

Vitamin E or the vitamin E-group is used as a collective term forfat-soluble, naturally occurring compounds having a chroman structureand a C₁₆ side chain (tocopherols). Tocopherols are chroman-6-ols(3,4-di-hydro-2H-1-benzopyran-6-ols) which are substituted in position 2with a 4,8,12-trimethyltridecyl residue. Tocopherols are slightlyyellowish-reddish oily liquids that are insoluble in water, yet solublein fats and oils, as well as in the usual solvents for fats. It isdifferentiated i.a. between α-, β-, γ-, δ-, and ε-tocopherols, thelatter still having the original unsaturated phenyl side chain, as wellas α-tocoquinones and hydroquinones in which the pyrane ring system isopened. The most frequent and most effective natural tocopherol is theα-tocopherol (2R, 4′R, 8′R)-form (trivial name: RRR-α-tocopherol) whichis synthetically accessible like all other stereoisomers (RömppChemie-Lexikon, 10^(th) Edition, pp. 4572/4573 and 4878-4886).

Originally described as an anti-sterility vitamin, today the opinionprevails that the vitamins of group E mainly act as a scavenger ofhyperoxide and peroxide radicals, as an antioxidant for unsaturatedfatty acids for LDL, for vitamin A and carotenes. Vitamin E is alsoattributed an important role in inhibiting inflammations and in theimmune function.

Therefore, vitamin E preparations are mainly used incirculation-stimulating and lipid-lowering agents for humans andanimals.

The invention has as its object to provide new medical applications forAfamin. Moreover, vitamin E preparations having an improved activity, inparticular as regards their antioxidant properties, are to be provided.

According to the invention, this object is achieved by a preparationcontaining Afamin, in particular in combination with vitamin E, whichcan be used to produce a preparation for the treatment of oxidativestress, in particular for the treatment of neurodegenerative diseases.According to the invention, surprisingly it has been found that bycombining vitamin E and Afamin, a vitamin E preparation with improvedproperties can be provided, in particular a vitamin E preparation havingan increased antioxidant action.

Even though a number of the most varying functions has been postulatedfor Afamin (primarily as a transporting system for fatty acids,hormones, enzymes, dyes, trace metals and medicaments, as well as adirect antioxidant (cf. WO 95/27059)), it has additionally shownaccording to the invention that Afamin itself (even without vitamin E)has a neuroprotective function which is increased by a surprisingsynergistic effect with vitamin E. Accordingly, primarily thecombination preparation of Afamin and vitamin E according to theinvention can be provided for the treatment of oxidative stress for awide range of applications. According to the invention, it has beenshown that the combination of Afamin and vitamin E is particularly wellsuited for the treatment of neurodegenerative diseases which areassociated with oxidative stress.

This has been surprising insofar as up to now it has not been possibleto confirm the previously postulated applications for Afamin (cf. WO95/27059, in which i.a. the use for rheumatoid arthritis, ARDS, sepsis,arteriosclerosis etc. have been considered possible, yet without beingcapable of providing proof of the same or presenting plausible modeltest). It has been the more unexpected that Afamin, primarily incombination with vitamin E (and not, e.g., in combination with vitaminD, a known binding partner of another protein from the albumin group)exhibits a significantly improved antioxidant effect, as compared to theindividual components.

By vitamin E, according to the invention, all the natural and syntheticbiologically active tocopherol preparations as initially described areto be understood, in particular tocopherol preparations derived fromnatural sources, in particular from vegetable oils, such as, e.g., soy,wheat, maize, rice, cotton, alfalfa and nuts, as well as from fruit andvegetable, e.g. raspberries, beans, peas, fennel, bell peppers, blacksalsifies and celery. Also the synthetically produced tocopherolpreparations, including the derivatives thereof, such as, e.g.,tocopheryl acetate, -succinate, -nicotinate and-poly(oxyethylene)-succinate are usable within the scope of the presentinvention (cf. Römpp Chemie-Lexikon, pp. 4572/4573).

By Afamin, according to the invention all polypeptides which have abiological activity in common with the natural Afamin are to beunderstood, in particular various glycosylated, deglycosylated,non-glycosylated, lipidated or de-lipidated forms, as well as variantsof Afamin prepared by recombinant technologies. A detailed structuraldefinition of Afamin is, e.g., given in WO 95/27059 which is expresslyincluded herein by reference thereto. In addition, recombinantly orproteolytically produced isolated sub-domains as well as chemicallyderivatized or modified forms (e.g., by acetylation, e.g. on lysine sidechains) and recombinantly or chemically produced extended versions ofthe protein, e.g. by attaching functional peptides which enhance thecrossing of the blood-brain barrier (as described, e.g., in Mazel et al.(Anticancer Drugs 12(2) (2001), 107-116), Temsamani et al. (Pharm. Sci.Technol. today 3(5) (2000), 155-162), Rousselle et al. (Mol. Pharmacol.57(4) (2000) 679-6866), Rousselle et al. (J. Pharmacol. Exp. Ther.296(1) (2001), 124-131) included.

Free radicals are involved in a number of acute and chronic neurologicaldisorders, in particular in focal ischaemia, trauma, epilepsy,Huntington's Disease, Alzheimer's Disease, amyotropic lateral sclerosis(ALS), AIDS, dementia and other neurodegenerative diseases. Furthermore,there exists an increasing number of examinations which suggest aparticipation of reactive oxygen species (ROS) in traumatic braininjuries. Also the impaired sensitivity of various receptor systems withincreasing age indicates an increasing deterioration of the reactions tooxidative stress.

Glutamate-induced cytotoxicity is a useful model system for testingcompounds as regards their antioxidant activity (Kabayashi et al., FreeRadic Res., 32 (2000), 115-124). Glutamate-induced cytotoxicity inHt-4neuronal cells has been attributed to oxidative stress caused by thereduction of cellular glutathione. Glutamate induces apoptoses incortical rat neurons and in the hippocampus-HT-22 cell line from mice byblocking the cystein uptake, whereby intracellular glutathione isreduced. This in turn results in the enrichment of ROS. A lowconcentration of α-tocopherols proved to be highly effective to protectthe neuronal cells against cytotoxicity.

Neuropathologies of Huntington's Disease are caused by an excessiveactivation of glutamate-coupled ion channels, whereby neurons are killedby oxidative stress.

α-Tocopherol might prevent oxiradical damage of the cell membrane, and aslowing down of the course of Huntington's Disease has been discussedfor such preparations. In particular, an antioxidant therapy might slowdown the rate of reduction of the motor abilities in the course ofHuntington's Disease (Peyser et al., Am. J. Psychiatry, 152 (1995),1771-1775).

The expression of two mutant superoxide dismutases (SODs) associatedwith familial ALS cause the death of differentiated PC12 cells, uppercervical ganglia neurons and pyramidal neurons in the hyppocampus(Chadge et al., J. Neurosci, 17(22) (1997), 8756-8766). At cell death,many characteristics which are typical of apoptosis could be found. Thiscell death might be prevented by an efficient treatment with vitamin E(Chadge et al., J. Neurosci, 17(22) (1997), 8756-8766).

The striatum contains a high concentration of oxidatable dopamine. Anolder organism exhibits a reduced capability of answering to oxidativestress, whereby this region becomes very susceptible to damage caused byfree radicals. Dopamin neurons are especially susceptible to such damageand diseases. A loss of Dopamin-neurons is associated with Parkinson'sDisease, e.g., and in that instance harmful oxygen-free radicals areaccumulated. α-Tocopherols might protect cells against cytotoxic effectscaused by Dopamine and L-Dopa (Ebadi et al., Prog. Neurobiol. 48(1)(1996), 1-19).

There also exists convincing epidemiologic and in vitro evidence thatchronic oxidative stress occurs in persons afflicted with Down syndrome(Javanovic et al., Free Radic Res., 9 (1998), 1044-1048). Such patientsdevelop an Alzheimer-like change in the brain, starting at an age offrom 30 to 40 years. In in vitro studies it could be shown that thereduced viability of Down syndrome neurons can be changed byantioxidants, such as vitamin E. The uptake of such oxidants with thefood alone does not seem to cause a sufficient change, or improvement,respectively, as regards the biomarkers for oxidative stress.

Oxidative damage has also been associated with acute degenerativediseases, such as epilepsy, trauma and cerebral ischaemic conditions,such as, e.g., in strokes. In these neurological disorders, theantioxidant therapy has always been considered as suitable, yet so farefficient means therefor have been missing.

Haloperidol, a dopamine receptor antagonist, is often prescribed for thetreatment of schizophrenia and other affective disturbances. Oxidativestress is considered as one of the main clinical side effects ofhaloperidol. This compound is lethal for HT22 cells of mouse hippocampusin a concentration-dependent manner and causes the cell death byoxidative stress. HP-induced oxidative cell death can best be preventedby vitamin E preparations (Post et al., J. Neurosci, 18(20) (1998),8236-8246); Sagara, J. Neurochem. 71(3) (1998), 1002-1012). On molecularlevel, haloperidol specifically induces the activity of theRedox-sensitive transcription factor NF-κB. This intensified NF-κBactivity could be blocked by neuro-protective antioxidants.

Even though the effects of antioxidant enzymes are contradictory inAlzheimer's Disease, changes in the glutathione-peroxidase orsuperoxide-dismutase activities have been observed by many researchers,and there are many hints suggesting that oxidative stress also plays animportant role in Alzheimer patients. Above all, it could be shown thatsubstances which are capable of scavenging free radicals, such asvitamin E, Selegilin and ginkgo biloba extract Egb 761, exhibit apositive effect in the therapeutic treatment of Alzheimer patients(Christen, Am. J. Clin. Nutr., 71(2) (2000), 621s-629s).

For these reasons, the Afamin according to the invention, particularlythe combination preparation of Afamin and vitamin E, preferably isemployed for the treatment of Alzheimer's Disease, Huntington's Diseaseor the amyotropic lateral sclerosis.

The combination preparation according to the invention thus isparticularly useful for the treatment of neurodegenerative dementias,such as they occur with these diseases, e.g.

According to a further embodiment, the present invention relates to theuse of Afamin, in particular in combination with vitamin E, forproducing a preparation for the treatment of acute neurodegenerativediseases, in particular for the treatment of epilepsy, trauma andcerebral ischaemia (a stroke, e.g.).

For the reasons set out above, Afamin, or the combination preparationaccording to the invention, respectively, is also suitable for thetreatment of Parkinson's Disease or for the treatment of the changesoccurring in patients afflicted with Down syndrome, which changes aresimilar to Alzheimer's Disease.

According to another aspect of the present invention, also oxidativestress occurring as a side effect of a medicament treatment can beprevented with Afamin, or with the combination preparation according tothe invention. In doing so, the Afamin, or the combination preparationaccording to the invention, can be provided together with the respectivemedicament, or it may be administered separately.

For all these indications, a positive therapeutic or prophylactic effecthas already been described or at least plausibly been postulated by theexperts in this field. With the combination preparation according to theinvention with which the antioxidant effect of vitamin E preparations isdecisively improved, according to the invention these indications can betreated or prevented more efficiently or at least their progress can beretarded.

Afamin, or the combination preparation according to the invention can beused for all these diseases both therapeutically and prophylactically,whereby the onset of the classically found symptoms can be prevented, orthe course of the disease is retarded, respectively. Accordingly, thepresent invention also relates to the use of Afamin, or of the inventivecombination preparation, respectively, for producing a neuroprotectivepreparation.

In a further aspect, the present invention relates to the use of Afamin,in particular in combination with vitamin E, for producing a preparationfor the treatment of infertility disorders. According to the invention,it has been shown that infertility disorders can efficiently be treatedwith the help of Afamin. Furthermore, vitamin E, which originally hasbecome known as a fertility factor, can decisively be improved in itsper se known fertility effect by a combination with Afamin.

According to a further aspect, the present invention relates to avitamin E preparation having an increased antioxidant effect, which ischaracterized in that it contains Afamin in addition to a vitamin Epreparation produced from synthetic or natural sources. Preferably,Afamin and vitamin E have a molar ratio of between 20 to 100 vitaminE/Afamin. This ratio takes into consideration the theoretical maximumloading of an Afamin molecule by 18 molecules of vitamin E (cf. FIG. 3).In particular, Afamin can be used which is derived from recombinantsources, which may differ from natural, human Afamin e.g. in its mode ofglycosylation, its lipid content or even in its amino acid sequence.

Besides the usual pharmaceutical carrier substances, also furtherpharmaceutically effective components may be admixed to the preparationaccording to the invention, e.g. components which are already known fora certain indication.

The invention will now be explained in more detail by way of thefollowing examples and the drawing figures to which, of course, it shallnot be restricted. Therein,

FIG. 1 shows the glutamine synthetase activity after a 5-day treatmentwith 3-NP in the presence of various protective substances;

FIG. 2 a shows Afamin and the combination of Afamin with vitamin E whichprotects against neuronal cell death, triggered by partial serumwithdrawal to 2%;

FIG. 2 b shows Afamin and the combination of Afamin with vitamin E whichprotects against neuronal cell death, triggered by 100 μM H₂O₂;

FIG. 2 c shows Afamin and the combination of Afamin with vitamin E whichprotects against neuronal cell death, triggered by 20 μM pre-aggregatedbeta-amyloid peptide;

FIG. 3 shows a Scatchard-plot analysis of the binding of vitamin E toAfamin.

EXAMPLES Example 1 3 NP(3-nitropropionic acid)-induced Reduction ofGlutamine Synthetase (GS) Activity

Because of the mechanistic and pathological similarities between 3-NPlesions and Hungington's Disease (HD), 3-NP has been proposed as analternative HD-model. Malonate and 3-NP are inhibitors of the succinatedehydrogenase which produce an energy loss and lesions which are similarto those observed in HD. Systemic administration of 3-NP causes aprogressive locomotory deterioration which corresponds to that of HD.3-NP causes a highly selective striatal degeneration. It differsmechanistically from excito-toxic lesions in that 3-NP irreversiblyinhibits the mitochondrial citrate cycle and leads to reduced ATP valuesand increased lactate concentrations.

Assay:

After the extraction of brain spheroids and a one-week culturing,D,L-alpha-tocopherol (0.1 mM) were added to the medium. Two weeks oldspheroids were plated in a plate having six wells. Shortly after theexposure, the medium was replaced by 2 ml of fresh medium. The spheroidswere exposed to 3-NP (0.5 and 5 mM, pH adjusted to 7 to 8), or to theprotectors vitamin E (0.1 mM), glutathione (1 mM), DTT (0.25 mM), L-NAME(N(G)-nitro-L-alanine-methylester) (0.1 mM), Afamin (without vitamin E(0.3 and 3 μg/ml) or combinations of 3-NP with the protectors for 5days. After 2 days, freshly prepared 3-NP and protectors were added tothe medium (Mathews et al., J. Neurosci, 18(1) (1998), 156-163);Borlongan et al., Neurosci Biohav. Res. 21(3) (1997), 289-293).

The results are illustrated in FIG. 1. Here it is shown that Afamin hadby far the highest effect of all the tested substances in protecting theglutamine synthetase (GS, an astrocytic enzyme which is highly sensitiveto oxidative stress) against inactivation by 3NP. As mentioned before,this system with 3NP constitutes a recognized model for excitotoxicityand serves as a model for Huntington's Disease.

Example 2 Neuroprotective Effect in Isolated Cortical Neurons of ChickenEmbryos

The neuroprotective effect of Afamin was examined by assaying isolatedcortical neurons of chicken embryos. The effect of Afamin was tested ina low-serum assay and in two lesion assays, namely in a β-amyloid and aH₂O₂-lesion assay.

After the low serum assay or the lesion assay, the MTT assay is used tocheck the viability of neuronal cells(MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltriazolium bromide). Thisis a suitable model which is extremely reproducible. The test alsoallows for assaying a large number of samples, and dose-dependentdifferences in the activity of a substance can be evaluated.

The neuronal viability of cells in vitro can be determined by means ofthis calorimetric MTT test. The MTT assay is based on the metabolicreduction of MTT, a substance of yellow color, to dark-blue formazanecrystals by mitochondrial dehydrogenases (e.g. succinate dehydrogenase).The crystals are dissolved, and the spectrometric absorption is measuredat a suitable wave length. It could be demonstrated that dead cells arenot capable of cleaving MTT; dormant cells produce less formazan,therefore this assay can be used for quantifying the viability of cells,since this reaction is only catalyzed by live active cells (Mosmann etal., J. Immunol. Meth. 65(1983), 55; Bernabei et al., Hemat. Oncol. 7(1989), 243; Barltrop et al., Bioorg. & Med. Chem. Lett. 1 (1991), 611;Cory et al., Cancer Commun. 3 (1991), 207).

In detail, isolated cortical neurons of 8- (lesion assays) or 9- (lowserum assay) day-old chicken embryos (White Leghorn or Lohman BrownHybrid strain) were used.

The neurons were prepared by breaking ethanoltreated eggs and puttingthe embryos into a plastic dish. After decapitation, the hemisphereswere removed and collected. Loose tissue and meningial membranes wereremoved, and the hemispheres were mechanically dissociated.

80 μl cell suspension containing 6×10⁵ cells/ml nutrient medium wereadded to each well of the microtiter plate which already contained 80 μlof medium with or without the substances. The plates were kept at 37°C., 95% humidity and 5% CO₂ without a change of media (up to 8 DIV).

The low serum medium contains 100 ml of DEMEM with 1 g of glucose/l, 2%fetal calf serum, 0.01% Gentamycin and 2 mM L-glutamine. The nutrientmedium for the lesion assays contains 100 ml of DMEM with 4.5 g ofglucose/l, 5% fetal calf serum, 0.01% gentamycin and 2 mM L-glutamine.

At DIV 8, the cells were treated with 20 μM of pre-aggregated β-amyloidpeptide (Aβ₂₅₋₃₅; Sigma) for 72 hours, or with 100 μM H₂O₂ for 24 h.

At the end of each experiment, the viability of the cultures was testedby means of the MTT assay with a plate reader at 570 nm.

The results are illustrated in FIGS. 2 a-c and show that Afamin, vitaminE and, above all, the combination of these two substances was capable ofreducing the apoptosis in 2% low serum medium (FIG. 2 a), and the damageby β-amyloid peptide or H₂O₂, respectively, which could be observed incontrol assays.

1. A method of treating Alzheimer's disease comprising: obtainingAfamin; and administering an effective amount of the Afamin to a subjecthaving Alzheimer's disease.
 2. The method of claim 1, further comprisingadministering vitamin E to the subject.
 3. The method of claim 2,wherein the Afamin and vitamin E are combined prior to administration.4. The method of claim 1, wherein the Afamin is further defined asrecombinant Afamin.